Small numbers of anti-host alloreactive T cells contained in the donor stem cell product are responsible for GVHD. Immunosuppression and global T cell depletion not only reduce the incidence and severity of GVHD but also non-specifically reduce the host's immune competence to pathogens and cancer. An alternative approach to controlling donor anti-host alloreactivity is to attempt to inactivate or deplete only the donor anti-host alioreactive T cells leaving the remainder of the T cell repertoire intact. We have basic, pro-clinical and clinical data supporting this concept. Two Phase I trials of haploidentical HSCT have been conducted where donor anti-host alloreactive T cells contained in the donor bone marrow were anergized ex vivo through alloantigen presentation and blockade of the B7:CD28 costimulatory pathway. These studies have demonstrated feasibility, tolerability, less than expected rates of GVHD and no late infectious problems. However, alloreactivity was incompletely controlled and we have yet to demonstrate that non-alloreactive T cell repertoire is retained and functional. Project 6 is committed to developing strategies to improve the success and exportability of this approach. Our working hypothesis is that a mechanistic understanding of molecular pathways that underlie the induction and maintenance of alloantigen specific T cell anergy will provide us with strategies to improve clinical interventions to control donor anti-host alloreactivity while retaining adoptively transferred immunity to pathogens and potentially cancer. Aim 1 proposes to undertake clinical trials to evaluate the capacity of adoptively transferred donor T cells anergized to host alloantigens to rapidly reconstitute a broad functional T cell repertoire. Aim 2 proposes to attempt to improve control of donor anti-host alloreactivity ex vivo by manipulating additional pathways invoNed in the induction and maintenance of anergy. Aim 3 proposes to determine which of the ex vivo approaches to control alloreactivity developed in Specific Aim 2 maximize retention of the pathogen specific repertoire and function. These 3 Aims are highly focused and interactive. Aim 1 will immediately embark upon a clinical trial to adoptively transfer anergized donor PBMC on day 35 following a "Perugia" haploidentical HSCT. Both GVHD and reconstitution of a broad T cell repertoire are the major endpoints. Aim 2 will simultaneously attempt to modulate additional pathways to improve the induction of anergy. These approaches may replace or be added to B7 mediated costimulatory blockade. Aim 2 provides the translational platform upon which our next clinical trials will be based. Finally, Aim 3 is responsible for retention of pathogen and tumor specific immunity. It proposes to develop strategies to identify and characterize anergic T cells and to study anergization strategies in vitro and in patients post HSCT to optimize the success of our clinical studies. Project 6 is interactive with all Projects that will be undertaking clinical translation (4, 5, and 7) and is highly dependent upon all Cores for its success.